Technical Abstract: The objective was to determine cholesterol concentration and fatty acid weight percentages in longissimus dorsi (LD) and semitendinosus (ST) muscles of Hereford (normal muscling, H), Limousin (moderate muscling, L), and Piedmontese (muscular hypertrophy, P) crossbred cattle. Sires (12 per breed) were bred to crossbred cows at random to produce F1 calves that were inter se mated within sire breed to produce F2 calves. P-cross calves were genotyped for the G-A transition mutation at the myostatin locus characteristic of P, and their genotypes were classified on of having 0 (P0), 1 (P1), or 2 (P2) copies of the mutant allele (mhP). Saturated fatty acids were greatest in H and L in LD (P=0.05, 42.8%) and ST (P=0.04, 38.5%). Monounsaturated fatty acids were lowest (P<0.01) in P2 for each muscle (34.5% in ST; 36.1% in LD). Polyunsaturated fatty acids (PUFA) were highest (P<0.01) in P2 LD (10.6%) compared with the other genotypes (4.8%). In ST, PUFA were highest (P<0.01) in P2 (12.7%), lowest (P<0.01) in H (5.8%), and intermediate in L, PO, and P1 (7.6%). In LD, omega-3 were highest (P<0.01) in P2 (1.4%) and lowest in PO (0.5%). Weight percentage of total conjugated linoleic acid was not affected by genotype in LD (0.2%, P=0.25), or ST (0.2%, P=0.28). Cholesterol concentration was not affected by genotype in LD (50.5 mg/100 g, P=0.44). In ST, cholesterol concentration was highest (P=0.02) in P2 (54.6 mg/100 g), and similar for the other genotypes (51.1 mg/100 g). We conclude that P2 contain higher ST muscle cholesterol concentrations. Weight percentages of PUFA, in particular the omega-3 fatty acids, were consistently higher in P2.